Brucella infects a significant number of people and domestic herds in developing nations and domestic and wild animals of the United States. Brucellosis in man may be caused by a number of strains of Brucella including B. abortus (cattle), B. suis (hogs), B. rangiferi (caribou), B. melitensis (sheep and goats) and B. canis (dogs). The disease is acquired by exposure to secretions and excretions of infected animals and by ingestion of milk or milk products from infected animals. The current live-attenuated vaccine strains for animals cause disease in humans, abortions in adult animals and complicates immunological screening for infected animals, since they stimulate production of antibodies to lippopolysaccharide (LPS) which is detected in the milk and serum of vaccinated animals. These antibodies are detected in serological tests used to screen milk and meat for Brucella.
Two approved live-attenuated Brucella vaccines are currently used to vaccinate animals. Strain 19 is currently used to vaccinate animals against B. abortus infections. This live-attenuated strain has several characteristics that make it undesirable as a human vaccine. The attenuating mutation in this strain is unknown; it sero-converts vaccinated individuals making differentiation of infected and vaccinated individuals difficult and causes significant disease in humans. Another live-attenuated veterinary vaccine B. melitensis strain, REV1 has the same limitations as Strain 19. Recently, a rough mutation of B. abortus, RB51, has been tested and found to be safe and protective in a number of different animal models. This rough live-attenuated strain was derived from a rifampin resistant strain and the mutation causing the rough phenotype and attenuation is not identified. Although RB51 does not stimulate the production of antibodies to LPS, it stimulates portective immunity in animals and does not cause abortions in vaccinated animals. Since the nature of the genetic defect causing the rough phenotype is not known, it is not possible to predictably make a vaccine under appropriate manufacturing proceedures for human use. Brucella strains have not been used as live-attenuated vaccine carriers for recombinant proteins.
Brucella also presents a potential biowarfare threat for military personnel. Multiple antibiotic resistant strains of Brucella have been constructed by others and pose a significant threat to the morbidity and mortality of infected personnel.
O'Callaghan (Infection and Immunity, Vol. 56, No. 2, pp 419-423 (1988)) describes deletion mutants of Salmonella having purE and purA deletions. Neither was effective as an oral vaccine. Hence, O'Callaghan would direct interest of those of ordinary skill from the purE deletion mutants for use as vaccines.
Houng, et al, (Abstracts of the 94th General Meeting of ASM, page 133, Abstract D-208) teaches that the writers identified purEK genes and identified two polypeptides that are products of these genes. However, they do not teach any particular mutant, nor do they teach how to make any mutant. Furthermore, they do not suggest any particular use for any mutant. (Taken with O'Callaghan, one of ordinary skill would not be directed to use such a mutant for a vaccine.)
Smith and Heffron (Infection and Immunity, Vol. 55, No. 11, pp 2774-2776 (1987)) teaches transposon Tn5 mutagenesis of Brucella abortus involving transposon Tn5. His construct is not made by the method of the invention. The instantly claimed invention does not involve introduction of Tn5 or Tn5 lac into a DNA sequence. Adams, in Advances in Brucellosis Research, Chapter 17, pages 250 to 276 (1990) suggests that Tn10 mutagenesis may be used to identify genes encoding virulence factors for adherence, invasion and intracellular survival, and that this method might be applicable to study of Brucella. He suggests no particular mutation. The instantly claimed invention was not suggested by Adams and was not made by the methods of Adams. Stocker in U.S. Pat. No. 4,735,801, suggests similar means of making mutants of Salmonella strains. Stocker introduced one or more modifications in biosynthetic pathways for products unlikely to be found in the disease susceptible host. Auxotrophic mutations are produced in one or more pathways by deletion or inversion of the target gene. These mutations can not be repaired by a single step and are referred to as non-reverting. In this patent he identifies aro, dap, pab and pur target genes for the construction of live vaccine strains. The pur gene targets claimed were those biosynthetic genes involved in the conversion of inosine monophosphate to adenine. He does not suggest the deletion mutants of the invention.